Austrofundulus limnaeus is a species of annual killifish which inhabits ephemeral ponds in South America. The species is able to survive seasonally desiccating ponds due to their ability to produce robust embryos. The embryos of this species are capable of entering a developmental arrest, termed diapause II, which precedes the onset of drought. While in this arrested state embryos exhibit the greatest tolerance to anoxia of any characterized vertebrate at 25ºC. Furthermore, when raised at 30ºC, embryos escape the entrance to diapause II and go on to develop directly. Currently, little is known about the molecular mechanisms which induce and maintain this developmentally arrested state. In this study I have developed methods to analyze changes in histone modifications in the context of diapause II. Histone modifications were chosen due to their extreme conservation and well characterized role as modulators of gene expression in other systems. Results utilizing adapted immunobased assays show significant changes in the global amount of H3S10P, H3K27me and H3K4me, as the embryos progress from early embryogenesis through the exit of diapause. Additionally, it is revealed that there exists a degree of phenotypic plasticity with regards to the entrance into diapause II which is modulated by the environment …

The purpose of this study was to examine the hormonal response to acute ethanol ingestion following a bout of heavy resistance exercise in women. Eight resistance trained women completed two identical acute heavy resistance exercise tasks (AHRET). From 10-20 minutes post-AHRET, participants consumed either a grain ethanol or a placebo beverage. Blood was collected before (PRE) and immediately after the AHRET (IP) and then every 20 minutes for five hours. Blood collected after beverage ingestion was pooled into 3 batches (phases: 20-40 minutes, 60-120 minutes, and 140-300 minutes post-exercise) and analyzed for serum total testosterone (TT), free testosterone (FT), insulin-like growth factor-I (IGF-I), human growth hormone (GH), cortisol (COR), and estradiol (E2) concentrations. Circulating concentrations of TT were significantly greater at P20-40 than at PRE, P60-120, and P140-300. Circulating concentrations of FT were significantly greater at P20-40 than at all other times. Circulating concentrations of GH were significantly greater at IP than at PRE, P60-120, and P140-300. Circulating concentrations of COR were significantly greater at P20-40 than at all other times. Additionally, COR concentrations at P140-300 were significantly lower than at all other times. Circulating concentrations of IGF-1 were significantly greater at P20-40 than at P60-120 and P140-300. Circulating concentrations …

This study determined the effect of hypoxia on air-breathing onset and physiological and morphological characters in larvae of the air breathing fishes Trichopodus trichopterus and Betta splendens. Larvae were exposed intermittently (12/12 h daily) to 20, 17, and 14 kPa of PO2 from 1 to 40 days post-fertilization. Survival, onset of air breathing, wet body mass, O2, Pcrit were measured every 5 dpf. Hypoxia advanced by 4 days, and delayed by 9 days, the onset of air breathing in Betta and Trichopodus, respectively. Hypoxia increased larval body length, wet mass, and labyrinth organ respiratory surface of Betta, but did not affect these factors in Trichopodus. Hypoxic exposure increased O2 by 50-100% at each day throughout larval development in Betta, but had no effect on larval Trichopodus. Hypoxia decreased Pcrit in Betta by 37%, but increased Pcrit in Trichopodus by 70%. Larval Betta reared in hypoxia showed a modified heart rate:opercular rate ratio (3:1 to 2:1), but these changes did not occur in Trichopodus. Compared to Betta, the blood of Trichopodus had a higher P50 and much smaller Bohr and Root effects. These interspecific differences are likely due to ecophysiological differences: Betta is a non- obligatory air-breather after 36 dpf with …

Sirtuins (SIRT) regulate the transcription of various genes involved in the development of diet-induced obesity and chronic disease; however, it is unknown how they change acutely following a high-fat meal. The purpose of this study was to determine the effect of a high-fat meal (65% kcals/d; 85% fat recommendation), on SIRT1-7 mRNA expression in blood leukocytes at 1, 3, and 5-h post-prandial. Men and women (N=24) reported to the lab following an overnight fast (>12H). Total RNA was isolated and reverse transcribed prior to using a Taqman qPCR technique with 18S rRNA as a normalizer to determine SIRT1-7 mRNA expression. An additional aliquot of serum was used to measure triglycerides. Data was analyzed using a RM ANOVA with P<0.05. Triglycerides (P<0.001; 124%) peaked at 3-h. SIRT 1 (P=0.004; 70%), and SIRT 6 (P=0.017; 53%) decreased expression at 3-h. SIRT4 (P=0.024) peaked at 5H relative to baseline (70%) and 3-h (68%). To our knowledge, this is the first study to report that consumption of a high-fat meal transiently alters SIRT mRNA expression consistent in a pattern that mirrors changes in serum triglycerides. Decrease in expression of SIRT1 and SIRT6 combined with an increased SIRT4 would be consistent with an increase in …

N-Acylethanolamines (NAEs) are fatty acid derivatives in plants that negatively influence seedling growth. N-Lauroylethanolamine (NAE 12:0), one type of NAE, inhibits root length, increases radial swelling of root tips and reduces root hair numbers in a dose dependent manner in Arabidopis thaliana L. (ecotype Columbia). A forward genetics approach was employed by screening a population of T-DNA “activation-tagged” developed by the Salk Institute lines for NAE resistance to identify potential genes involved in NAE signaling events in Arabidopsis thaliana L. (ecotype Columbia). Seeds of the activation tagged lines were grown at 0, 25, 30, 50, 75 and 100 µM N-lauroylethanolamime (NAE 12:0). Ten plants which displayed NAE tolerance (NRA) seedling phenotypes, compared with wildtype (Columbia, Col-0) seedlings were identified. I focused on one mutant line, identified as NRA 25, where the tolerance to NAE 12:0 appears to be mediated by a single dominant, nuclear gene. Thermal asymmetric interlaced (TAIL) PCR identified the location of the T-DNA insert as 3.86 kbp upstream of the locus At1g68510. Quantitative PCR indicated that the transcript level corresponding to At1g68510 is upregulated approximately 20 fold in the mutant relative to wildtype. To determine whether the NAE tolerance in NRA 25 is associated with overexpression of …

Thauera aminoaromatica MZ1T, a floc-forming bacterium isolated from an industrial activated sludge wastewater treatment plant, overproduces exopolysaccharide (EPS) leading to viscous bulking. This phenomenon results in poor sludge settling and dewatering during the clarification process. To identify genes responsible for bacterial flocculation, a whole genome phenotypic sequencing technique was applied. Genomic DNA of MZ1T flocculation-deficient mutants were subjected to massively parallel sequencing. The resultant high-quality reads were assembled and compared to the reference genome of the wild type genome. We identified nine nonsynonymous mutations and one nonsense mutation putatively involved in EPS biosynthesis. Complementation of the nonsense mutation located in an EPS deacetylase gene restored the flocculating phenotype. The FTIR spectra of EPS isolated from the wild-type showed reduced C=O peak of the N-acetyl group at 1665 cm-1 as compared to the spectra of MZ1T floc-deficient mutant EPS, suggesting that the WT EPS was partially deacetylated. Gene expression analysis also demonstrated the deacetylase gene transcript increased before flocculation occurred. The results suggest that the deacetylation of MZ1T EPS is crucial for flocculation. The information obtained from this study will be useful for preventing viscous bulking and wastewater treatment system failure, and may have potential applications in the biotechnology sector for …

Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were …

Phenotypic plasticity is the ability of an organism to express different phenotypes in response to biotic or abiotic environmental cues. The ability of an organism to make changes during development to adjust to changes in its environment is a key to survival. Sexually reproducing organisms that have short life cycles and that are easy to raise in the laboratory are more conducive for developmental phenotypic plasticity. Considerable research has already been carried out on the brine shrimp, Artemia franciscana, regarding its morphology due to changing salinities. There is, however, little research considering subsequent generations and how there morphology might be affected by parental experiences. This study has examined: 1) the morphological effects of different rearing regimes of different salinity levels, and 2) the epigenetic transgenerational transfer of these morphological traits in A. franciscana. Measurements included rate of growth (as measured by instar), body size, body length, and other morphological traits. A gradual increase to more hyperosmotic conditions during development produced brine shrimp that were larger in size and also more developmentally advanced. Salinity stress experienced by adults had increased the growth rate in the F1 offspring of A. franciscana. Collectively, these data indicate that Artemia franciscana is a tractable model …

Cysteinyl leukotriene receptor 2, a G-protein coupled receptor known to be expressed and functional on human platelets. However, it seems that upon ligand activation the cysteinyl leukotriene receptor 2 activates a variety of signaling pathways in multiple cell types among different species. Previously, a former laboratory member Vrinda Kulkarni found cysteinyl leukotriene receptor 2 to be expressed on the surface of adult zebrafish thrombocytes. In this work I studied the characteristics of aggregation in adult zebrafish thrombocytes with the knockdown of cysteinyl leukotriene receptor 2. I used a newly developed knockdown method to study the function of cysteinyl leukotriene receptor 2. Knockdown of the cysteinyl leukotriene was confirmed using RT-PCR results showed p=.001, reduced sell surface level of expression of the cysteinyl leukotriene receptor 2 results showed that p=.002. I found that the knockdown of cysteinyl leukotriene receptor 2 results in prothrombotic thrombocytes by using flow cytometry p=.0001.

GPR17, a uracil nucleotide cysteinyl leukotriene receptor, belongs to the GPCR (G protein coupled receptor) family. It has been shown recently that inhibiting this protein in the nervous system in mice can lead to blockage of oligodendrocyte maturation, which supports myelin repair. Interestingly, our laboratory found GPR17 in thrombocytes. However, we do not know whether it has any function in thrombocyte aggregation or the nature of the ligand. In this paper, we studied the role of GPR17 in hemostasis, which is a fundamental defense mechanism in the event of injury. Using zebrafish as a model system, our laboratory has studied specifically thrombocytes, which play a significant role in hemostasis. The major reasons to use zebrafish as a model system are that their thrombocytes are functionally equivalent to human platelets, the adult fish are amenable to knockdown experiments, and they are readily available in the market. This study was performed by using a piggy back knockdown method where we used a chemical hybrid of control morpholino and an antisense oligonucleotide sequence leads to the degradation the mRNA for GPR17. After knockdown GPR17 in thrombocytes, the percent difference of the thrombocytes aggregation between the control and knockdown blood samples was measured by …

Natural flow regimes are critical for sustaining biodiversity and river integrity. Floods and droughts form an important component of river systems and control population sizes and species diversity across space and time. Modification of flow regimes, including disruption of the timing, magnitude and duration of flooding, is a global problem, and many new impoundments are planned for large river-floodplain ecosystems in the tropics. Flow modifications may cause dramatic non-linear responses in population sizes and have lasting effects through time, but such topics are poorly investigated over multi-year scales, especially in highly diverse tropical ecosystems. Using a long-term dataset from the Upper Paraná River floodplain, Brazil, I tested for threshold and legacy effects of fish assemblages to flood attributes, such as timing, magnitude, duration, rate of change and variation. Specifically, I hypothesized that long duration, high magnitude floods would elicit threshold responses in long-distance migratory fish species and these responses result in significant legacy effects detectable over multiple years. Consistent positive threshold responses to increasing flood duration and magnitude were detected for many species and not significantly correlated with reproductive guilds. Legacy effects were prevalent (i.e. identified for more than 90% of species) and including flood attributes from previous years increased …